Production of Rhizopus oryzae lipase using optimized Yarrowia lipolytica expression system

Abstract Yarrowia lipolytica is an alternative yeast for heterologous protein production. Based on auto-cloning vectors, a set of 18 chromogenic cloning vectors was developed, each containing one of the excisable auxotrophic selective markers URA3ex, LYS5ex, and LEU2ex, and one of six different promoters: the constitutive pTEF, the phase dependent hybrid pHp4d, and the erythritol-inducible promoters from pEYK1 and pEYL1 derivatives. These vectors allowed to increase the speed of cloning of the gene of interest. In parallel, an improved new rProt recipient strain JMY8647 was developed by abolishing filamentation and introducing an auxotrophy for lysine (Lys−), providing an additional marker for genetic engineering. Using this cloning strategy, the optimal targeting sequence for Rhizopus oryzae ROL lipase secretion was determined. Among the eight targeting sequences, the SP6 signal sequence resulted in a 23% improvement in the lipase activity compared to that obtained with the wild-type ROL signal sequence. Higher specific lipase activities were obtained using hybrid erythritol-inducible promoters pHU8EYK and pEYL1-5AB, 1.9 and 2.2 times, respectively, when compared with the constitutive pTEF promoter. Two copy strains produce a 3.3 fold increase in lipase activity over the pTEF monocopy strain (266.7 versus 79.7 mU/mg).


Introduction
The oleaginous yeast Yarrowia lipolytica (Y. lipolytica) has recently r eceiv ed incr eased attention for white biotec hnology a pplications, including as an alternative host for recombinant protein (rProt) production (for recent review see (Madzak 2021 )).
While the methylotrophic y east K omagataella phaffii (formerly Pichia pastoris ) has been extensively used as a platform for rProt pr oduction (for r ecent r e vie w see (Carneir o et al. 2022 )), Y. lipol ytica was also shown to be an attr activ e host for rProt production. The expression system developed at INRAE for Y. lipolytica has impr ov ed ov er the last 20 years (Nicaud et al. 2002, Madzak et al. 2004, Madzak 2015, 2021. Ev en though Y. lipol ytica is used less frequently than K. phaffii , it has been shown to be a more efficient host for se v er al pr oteins suc h as the pr oduction of Candida antarctica lipase B (CalB) (Theron et al. 2020 ).
Wild-type Y. lipolytica strains are often found on protein and lipid rich media. The yeast is well-adapted for this environment, due to the large number of genes coding for proteases and lipases. The main proteases are alkaline extracellular protease (Aep encoded by XPR2 ) and acid extr acellular pr otease (Axp1 encoded by AXP1 ), and the main lipases are encoded by LIP2 , LIP7 and LIP8 (Fickers et al. 2005, Fickers et al. 2011. The structure of the prepr o r egions and matur ation pr ocess of the Aep and Lip2 proteins pr esents a pr e-sequence of 13 amino acid (AA), a str etc h of dipeptide XA/XP of 10 units and 4 units, r espectiv el y, pr ocessed by a specific aminopeptidase and follo w ed b y a pr o-r egion of 27 AA and 12 AA, r espectiv el y, including a dimotif KR r ecognized by endopr otease Xpr6 (K ex2 homolog), follo wing b y the matur e pr otein (Celi ńska et al. 2018 ). The targeting sequences (signal sequence) and pro region of these proteins have often been used for heterologous pr otein secr etion (Madzak et al. 2004, Celi ńska et al. 2018, Celi ńska and Nicaud 2019. Se v er al host str ains hav e been de v eloped at INRAE for rPr ot pr oduction, deriv ed fr om the Fr enc h wild-type str ain W29 (Fig. 1 ). The first strain, Po1d, contains the deletion of the XPR2 gene coding for the alkaline extracellular protease Aep ( xpr2-322 ) (Nicaud et al. 2002 ). It also contains two non-r e v erting gene deletions of the URA3 gene and the LEU2 gene ( ura3-302 and leu2-270 alleles, r espectiv el y). Ther eafter, the MTLY60 str ain w as constructed b y successive gene deletion in order to delete the genes coding for the three main lipases, Lip2, Lip7 and Lip8, resulting in a host strain for lipase ov er expr ession (Fic k ers et al. 2005 ). A deri vati ve of this str ain, JMY1212, was cr eated by intr oducing a zeta doc king platform to allow the targeted integration of a unique copy of any zeta-based expr ession cassette. Suc h expr ession cassettes could be released from the auto-cloning vector of JMP62 type (Fig. 2 A) upon Not I digestion, resulting in an expression cassette devoid of any bacterial sequence . T his expression system is used for the scr eening of ne w enzymes and enzymes with impr ov ed enzymatic activity or thermostability , Emond et al. 2010, Bordes et al. 2011. Mor e r ecentl y, str ain JMY7126 was designed to tak e ad v anta ge of the ne w erythritol-inducible pr omoters, by Figure 1. Genealogy of Y. lipolytica main strains used for rProt production. All strains derived from the wild-type strain W29 (CLIB89). W29 wild-type strains and their deri vati ves: genotypes and phenotypes are indicated under each strain. Genetic modifications are indicated to the right of each strain. T he P o1D strain (Barth and Gaillardin 1996 ) contains first: the con v ersion of URA3 into ura3-302, corr esponding to ura3 ::pXPR2:SUC2, that is a 728 bp XhoI-EcoRV deletion in the URA3 coding region by inserting the S. cerevisiae SUC2 expressed under the XPR2 promoter, conferring the ability to grow on sucrose or molasses (Ura − ) (Nicaud et al. 1989 ); second: the leu2-270 corresponding to a 681 bp StuI deletion in the LEU2 coding region obtained by pop-in/pop-out method (Leu − ); third: the xpr2-322 corresponding to a 149 bp ApaI deletion in the XPR2 coding region, eliminating the alkaline extracellular protease Aep production (Aep − ). Thereafter, MTLY60 was obtained by successive gene deletion and marker rescue for the deletion of the lipase encoding genes LIP2 , LIP7 and LIP8, coding for the extracellular Lip2 (Pignède et al. 2000a ) and the membrane bound lipases Lip7 and Lip8 (Fickers et al. 2005 ) (Lip − ), according to (Fickers et al. 2003 ). The JMY1212 was obtained by insertion of the docking platform LEU2 -ZETA restoring the Leu + phenotype  ). The JMY7126 contained the deletion of the LYS5 gene coding for the sacc har opine dehydr ogenase introducing an additional auxotrophy for lysine (Lys − ), and the deletion of the erythrulose kinase EYK1 gene preventing erythritol degradation (Eyk − ) allowing efficient induction of gene expression when using erythritol-inducible promotors (Park et al. 2019b ). Finally, JMY8647 contained a mutation in the MHY1 gene eliminating strain filamentation (Fil − ).
deleting the erythrulose kinase encoded by EYK1 ( eyk1 ) and introducing an additional deletion of the LYS5 gene ( lys5 ) for multiple gene insertion (Soudier et al. 2019, Park et al. 2019a. Most of the vectors developed at INRAE for rProt production are based on the structure of the JMP62 auto-cloning vector (Fig. 2 A) and contain: a ZETA sequence for expression cassette integration into the genome, a Y. lipolytica marker flanked by I-Sce I for selecting transformants in Y . lipolytica , and one of a set of different promoters, introduced as a Cla I-Bam HI fragment, a terminator, and a region in which the Bam HI and Avr II restriction sites are used for cloning of the gene of interest. The Not I bacterial fragment contains the KanR marker for selecting E. coli transformants.
The main promoter used for rProt production is the strong constitutiv e pr omoter pTEF, used to select v ariants with impr ov ed properties , Emond et al. 2010, Beneyton et al. 2017 or to select the most efficient signal sequence for the rProt (Celi ńska et al. 2018 ). Lipid-inducible promoters have been de v eloped (pLIP2 and pPOX2) that could be induced by oleic acid (Nicaud et al. 2002, Sassi et al. 2016, ho w e v er they ar e impair ed by the oleic acid emulsion, making it difficult to measure growth. Mor e r ecentl y, erythritol-inducible pr omoters wer e designed and the EYK1 upstream activating sequence (UAS1 eyk1 ) was identified , Trassaert et al. 2017.
The identification of UAS from the XPR2 gene (UAS XPR2 ), from the TEF1 gene (UAS TEF ) and from the EYK1 gene (UAS EYK1 ), among others, and the generalization of the hybrid synthetic promoter a ppr oac h (Blazec k et al. 2013 ), has led to the de v elopment of hybrid promoters that allow the fine-tuning of gene expression by modulating this expression depending on the number of UAS repeats. Suc h str ategies wer e used in the de v elopment of str ong hybrid erythritol-inducible promoters (Trassaert et al. 2017, Park et al. 2019a ) and r ecentl y in de v elopment of bidirectional promoters for coordinated co-expression of two genes (Vidal et al. 2023 ).
To de v elop an effectiv e platform for rPr ot pr oduction, along with an efficient host and plasmid set, robust targeting sequences ar e r equir ed. Celi ńska and co w orkers hav e r eported the identification of robust signal peptides for protein secretion in Y. lipolytica and demonstrated that the expression level of a given heterologous protein was dependent on the signal peptide, and could be incr eased se v er al times by signal peptide selection, whic h e.g . r esulted in a 6 fold increase in production of a Sitophilus oryzae αamylase and a 4 fold increase in production of the Thermomyces lanuginosus glucoamylase (Celi ńska et al. 2018 ). The targeting sequences SP1, SP4 and SP6 were among those that sho w ed the best efficiency for both proteins and were selected for this study.
Lipases (EC 3.1.1.3), also called triglyceride hydrolases, are the most used enzymes to modify the structure of oils and fats. They ar e commonl y used to tailor natur al lipids to meet specific properties useful for food, nutrition, and cosmetic applications (Re yes-Re yes et al. 2022 ). Lipases are widely found in almost all kinds of living organisms in nature, mostly animals , plants , and microbes (Borrelli and Trono 2015 ). The majority of academic T hey contain a Cla I and Bam HI pr omoter r egion (indicated in r ed), a Bam HI and Avr II r egion for the expr ession of the gene of inter est (pur ple), a terminator (y ello w) and a Y. lipolytica mark er flank ed by I-Sce I restriction sites . T he gene conferring kanam ycin resistance (KanR) in E. coli and the mark er for the selection of Y. lipolytica tr ansformants ar e indicated by blue arr ows. Red arr ows r epr esent the zeta r egions for expr ession cassette integr ation. The expr ession cassette can be liberated by Not I digestion. (B) The newly developed chromogenic vector contains either LEU2 ex, LYS5 ex or URA3 ex as excisable marker and one of the six promoters. (C) Colors of E. coli strains containing the classical expression vector (white colonies) or producing RFP (red colonies) or AmilCP (blue colonies). (D) Typical transformation plate after gene cloning in an AmilCP expression vector or (E) in an RFP expression vector, enabling the identification of the colonies containing the recombinant plasmid (white colonies).
r esearc h about lipases and their industrial applications relies on se v er al commerciall y av ailable lipases, whic h mostl y (mor e than 50%) originate from and are produced by microorganisms, due to their gr eater r obustness, activity v arieties and yields (Bornsc heuer 2008, Fickers et al. 2011, Adlercreutz 2013, Rodrigues et al. 2013. Lipases from filamentous fungi families, such as Rhizopus oryzae (ROL), hav e been widel y used in the oil and fats industry, due to their high 1,3-regioselectivity to w ar d triglycerides that make them versatile in lipid modification (Rodrigues et al. 2013 ). The structur al featur es of one of the major nativ e lipases fr om Y. lipol ytica , Lip2 (Bordes et al. 2010 ), is very similar to ROL (Dong et al. 2022 ), making Y. lipolytica a potential excellent host for ROL production.
In this study, the Y. lipolytica expression platform was impr ov ed by increasing the rProt-using plasmid set with vectors producing c hr omopr oteins in E. coli, expanding the available selection markers for easier identification of r ecombinant expr ession v ectors, and allowing a faster cloning strategy. In addition, a new host str ain was gener ated, JMY8647, containing nine cum ulated gene deletions, unable to filament and containing two auxotrophic markers . T his new rProt platform was used for ROL production.

Strains and media
E. coli DH5 α (T hermoFisher Scientific , Les Ulis , F rance) w as used for plasmid pr opa gation. E. coli dam − /dcm − (Ne w England Biolabs, MA, USA) was used as a r ecipient str ain to pr e v ent Cla I methylation when r equir ed. All E. coli str ains used in this study are listed in Table 1 . The E. coli str ains wer e gr own at 37 • C in Lysogen y Br oth (LB) medium supplemented with either kanamycin sulfate (50 μg/mL) or ampicillin (100 μg/mL). Y. lipol ytica str ains built in this study are described in Table 2 . The genealogy of the main Y. lipolytica strains used for rProt production are depicted in Fig. 1 . For transformation and selection, Y. lipol ytica str ains wer e gr own at 28 • C in both rich medium (YPD) and minimal glucose medium (YNBD), prepared as described previously (Park et al. 2019a ). The YPD medium contained 10 g/L of yeast extract (Difco, Paris, France), 10 g/L of Peptone (Difco, Paris, France), and 10 g/L of glucose (Sigma Aldrich, Saint-Quentin Fallavier, France). The YNBD medium contained 1.7 g/L of yeast nitrogen base without amino acids and ammonium sulfate (YNBww; BD Difco, Paris, France), 5.0 g/L of NH 4 Cl, 50 mM phosphate buffer (pH 6.8), and 10 g/L of glucose. To meet the auxotrophic requirement, uracil (0.1 g/L) or lysine (0.8 g/L) were added to the culture media as necessary. Solid media were created by adding 1.5% agar. For rProt production, cells wer e gr own in the inducible medium YNBDE, containing 5 g/L of glucose and 5 g/L of erythritol for promoter induction. Cultures were performed in triplicates with 25 mL of YNBDE in 250 mL baffled flasks, at an initial optical density at 600 nm (OD 600 ) of 0.5 for 72 h at 28 • C, 160 r pm. Cultur es wer e centrifuged and supernatants were used for the evaluation of lipase production. Cell growth was followed by measuring the optical density at 600 nm (OD 600 ).  ( E. coli ). They contain the template plasmids or the ne wl y de v eloped acceptor c hr omogenic expr ession vectors . T he suffix '-ex' for the marker indicates the presence of Lo xR/Lo xP motifs that are excisable using a Cre-lox recombination method (Fickers et al. 2003

Chromogenic expression vector construction
The E. coli strains containing template plasmids and ne wl y dev eloped acceptor c hr omogenic expr ession plasmids ar e listed in Table 1 and depicted in Fig. 2 B. The donor plasmids were used for plasmid construction, to provide Cla I-Bam HI promoter fragment, I-Sce I excisable marker fr a gment, and Bam HI-Avr II fr a gment encoding c hr omogenic gene-the blue c hr omopr otein fr om cor al (AmilCP) or the Red Fluor escent Pr otein (RFP). The primer pairs used for plasmid parts amplification by PCR are described in Table  S1 (Supporting Information). The primer pairs were designed to introduce Cla I/ Bam HI or Bam HI/ Avr II restriction sites at the 5' and 3' ends of the amplified fr a gment, for pr omoter (pEYL1 and pEYL1-5AB) and gene (RFP) cloning, r espectiv el y. Restriction enzymes and T4 DNA ligase were obtained from NEB (MA, USA). PCR amplifications were performed using an Applied Biosystems 2720 Thermal Cycler, with Q5 ® High-Fidelity DNA Pol ymer ase (NEB) for amplification purposes and with GoTaq ® DNA Pol ymer ase (Pr omega, WI, USA) for construction verification. Restriction enzymes, ligase, and DNA pol ymer ases wer e used in accordance with the manufactur er's r ecommendations. Plasmids wer e isolated using a NucleoSpin Plasmid EasyPure Kit (Machery-Nagel, Duren, Germany), and digested fragments were purified using a NucleoSpin Gel and PCR Clean-up Kit (Mac hery-Na gel). The constructed plasmids were verified by digestion, by PCR and by sequencing. DNA sequencing was carried out by Eurofins Genomics (Ebersberg, German y). Benc hling softwar e was used for sequence analysis and primer design.

Protocol for gene cloning into chromogenic expression vectors
JMP62 destination vector (containing Kanamycin resistance) with blue or red chromogenic reporter and donor gene vector (containing Ampicillin resistance) were digested at 37 • C for 1 h and heat inactivated at 80 • C for 20 minutes . T he digestion products were mixed at a 1:5 ratio vector/insert for ligation at room temperature for 30 minutes. Competent E. coli cells were transformed with 10 μL of ligation mix and spread on LB kanamycin plates and incubated overnight at 37 • C. RFP and AmilCP were used as reporter genes for easy colored screening. White colonies were selected and verified by colony PCR. Plasmids containing the gene of interest were verified by sequencing. Typically, the 25 μL destination vector mix contained 0.5 μg of DNA, and 0.5 unit and 0.2 unit of thermo-inactivable Bam HI and Avr II restriction enzymes, r espectiv el y, (Thermo Fisher Scientific, Villebon sur Yvette, France) in Tango buffer. The 25 μL donor vector mix contained 1 μg of DNA, and 0.5 unit and 0.2 unit of thermo-inactivable Bam HI and Avr II restriction enzymes, respectively, in Tango buffer. The 20 μL ligation mix contained 50 ng of vector, 40 to 60 ng of donor

Vector construction for rhizopus oryzae lipase expression
Plasmids containing the codon optimized synthetic genes of the Rhizopus oryzae lipase (ROLop) with the different targeting sequence wer e pr ovided by BioCat (BioCat GmbH, Heidelber g, Germany) and cloned in pET-3a, as Bam HI-Avr II fragments (Table 3 ). They carry the ampicillin (AmpR) marker for selection in E. coli . The ROLop genes were assembled into destination vector JME5599 for SP comparison, then into JME5601, JME5602, and JME5579 for lipase expression with stronger or inducible promoters. After transformation in competent E. coli cells, plasmids containing the gene of inter est wer e v erified by PCR and sequencing (primers used are listed in Table S1, Supporting Information).

Construction of a new recipient strain for rProt production
The mhy1 deletion was introduced into JMY7126 using the CRISPR-Cas9 method described by (Larroude et al. 2020 ). To delete the MHY1 gene (YALI0B21582g), we targeted the sequence GGCGA CA GCA TGT AAA TGGG located at the beginning of the gene (162 bp downstream of the ATG). The guide sgRNA was introduced into the CRISPR-Cas9-LYS5 ex-platform re plicati ve vector by annealing two ov erla pping primer pairs sgRNA-MHY1-162 (Table S1, Supporting Information) that generated overhangs matching those of the BsmB I sites of the acceptor v ector, r esulting in the vector named GGE0440 (Table 1 ). Transformants were scr eened on selectiv e media (YNBD + ur acil) depending on their morphology; colonies that sho w ed no mark of filamentation on plate and in microscop y (Fig. 3 ) w ere selected. Sequencing of the MHY1 locus r e v ealed a 1 bp deletion at position + 160 bp from the ATG resulting in a frameshift ( Figure S1, Supporting Information). The strain was cured from the re plicati ve vector CRISPR-Cas9-LY S5 ex-sgRN A-MHY1 b y successiv e cultur e in ric h YPD media and the isolation of a Lys − clone . T his ne w str ain mh y1 w as named JMY8647 (Fil − ).

Construction of rhizopus oryzae (ROLop) expressing strain
The plasmids constructed for ROL expression were digested by Not I, which allo w ed the expression cassette to be released from Table 3. Expression plasmids used for ROL production ( E. coli strain).

Plasmid name Description Reference
This study the vector prior to JMY8647 tr ansformation. Tr ansformation of yeast cells used 400 ng of DNA and the lithium acetate method (Barth and Gaillardin 1996 ). Tr ansformants wer e selected on YNBD, YNBD + uracil, or YNBD + lysine medium based on their genotype. For each construction, 3 to 9 isolated transformants were selected as biological clones for further experiments (Table 2 ) and for eac h tr ansformant the integration of the expression cassette was verified by colony PCR with specific primers (Table  S1, Supporting Information). To construct pr ototr ophic str ains, the URA3 fr a gment fr om plasmid JME1046 and/or the LYS5 fr a gment from plasmid JME3265 were transformed. The prototrophic contr ol str ain (without ROL expr ession cassette) was named JMY8671 (pr ototr oph, eyk1, mhy1 ).

Lipase activity and growth onto lipid solid media
Drop tests were done using solid minimum YNBD media supplemented with 2% emulsified triolein (65% GC, Fluka Chemie A G , Germany) or shea olein containing more saturated fatty acid mainly including 1-palmito yl-2-oleo yl-3-stearo ylglycerol (POS) and 1-stearo yl-2,3-oleo ylglycerol (SOO) (AAK AB, Sweden). The solid minimum YNBD medium is described above and contained 20 g/L of glucose . T he trigl yceride stoc k em ulsions wer e sonicated three times for 1 minute follo w ed b y 1 minute resting on the ice in the presence of 0.5% Tween 40 (Sigma Aldrich, St Louis, USA) until the milky solution was obtained. The pr ecultur e was gr own overnight in YPD medium, the cells were centrifuged, washed, and resuspended at an OD 600 = 1 in liquid YNBD medium. A set of 4 of 10-fold dilutions of cell cultures was made (10 0 to 10 −3 respectiv el y), 3 μL of eac h dilution was plated onto the solid media plates . T he plates were incubated at 28 • C and screened for 5 da ys .

Lipase activity in culture supernatants
The activity in culture supernatants expressing R OL w as determined by measuring the velocity of releasing p -nitrophenol from 0.2 mM p -nitrophenol butyrate ( p -NPB) at pH 7.0, 30 • C. A stock solution of p -NPB was made using p NPB (Sigma Aldrich, St. Louis, USA) and 2propanol (Sigma-Aldrich, St. Louis, USA), mixed with a volume fraction of 0.15%. About 10 μL of supernatant from the 72 h culture was added into a 96well plate with 88 μL of 200 mM Na 2 HPO 4 /KH 2 PO 4 phosphate buffer, pH 7.0. The reaction was initiated by adding 2 μL of p NPB stock solution. The activity assay was monitored for 5 minutes, and the linear region was used to determine the lipase activity. One unit of lipase activity was set as the amount of enzyme that released 1 μmol p -nitrophenol per minute under the activity assay condition (U/mL). Specific activity was defined as units of lipase activity per mg of cell dry weight CD W (U/mg CDW ). The OD 600 /CD W correlation was measured as 1 unit of OD 600 corresponding to 0.11 mg of CDW. Experiments were performed in triplicate . T he standard error of the mean (SEM) was also determined.

Protein quantification
The protein concentration was quantified with Nanodrop TM 1000 (T hermo Fisher Scientific , Wilmington, USA) calibrated with a standard curve composed by a series concentration of bovine serum albumin (0.2 to 2.0 mg/mL).

Construction of new vector set for protein expression
For Y. lipolytica, we have previously developed expression vectorsso called 'auto-cloning vectors'-of JMP62 type as the integrating expression cassette is devoid of bacterial sequence (Pignède et al. 2000b, Nicaud et al. 2002 (Fig. 2 A). In order to generate an expanded set of plasmids useful for rProt production that could facilitate the cloning process, a gene conferring E. coli colored strain was inserted between the Bam HI and Avr II cloning sites (Fig. 2 B).
The plasmid set contains three different Y. lipolytica excisable markers: LEU2 ex, LYS5 ex or URA3 ex, and six different promoters: the str ong constitutiv e pTEF (Müller et al. 1998 ), the phasedependent hybrid Hp4d promoter (Madzak et al. 2000 ), and the hybrid erythritol-inducible promoters pHU8EYK, pEYK1-3AB (Park et al. 2019a ), pEYL1, and pEYL1-5AB (Vidal et al. 2023 ), conferring differ ent expr ession le v els upon erythritol induction. The plasmid set is listed in Table 4 . It contained 18 plasmids with six different promoters and three different markers. While the current JMP62 type plasmid did not contain c hr omogenic markers and did not give rise to colored E. coli transformants, the E. coli strains containing the ne w v ectors confer a red or a blue color when expressing the RFP or the AmilCP, r espectiv el y (Fig. 2 C). Upon transformation of the expression plasmids containing a gene of interest, E. coli transformants containing recombinant plasmids carrying the heterologous gene (white colonies) could be easily identified among the red or blue transformants containing the acceptor vector ( Fig. 2 D and E). Classical cloning r equir es the following steps: (i) acceptor vector digestion, (ii) dephosphorylation, (iii) a gar ose gel migr ation of the digested vectors (acceptor vector and donor vector containing the gene of interest), (iv) purification of the selected bands, (v) ligation, (vi) transformation of E. coli , (vii) selection of the transformants, and finally (viii) the screening of the correct expression vector to be used for Y. lipolytica transformation. This process requires about 1 to 2 weeks to be completed. Instead with the new method, the four initial steps could be performed at once. Indeed, both acceptor and donor v ectors ar e digested by the corresponding restriction enzymes ( Bam HI and Avr II), heat inactivated, and the mix used dir ectl y for ligation.
The efficiency of the cloning of the gene of interest using our method can be estimated by the ratio of white colonies to total colonies, as well as by the correct expression plasmid among the white colonies. To e v aluate the cloning efficiency, eight individual assemblies were performed with different ROL fragments (see belo w-section R OL cloning str ategies) and tr ansformed into E. coli . Typically, among about 30 to 120 kanamycin-resistant transformants , 58%-75% were white , demonstrating a mean of 66% cloning efficiency. Presence of the ROL in the recombinant plasmid was confirmed by E. coli colony PCR using a forward primer in the LYS5 marker and a r e v erse primer in the ROL gene (primer pair L YS5-internal2-F/ROL-internal-R, T able S1, Supporting Information). Among the white transformants, depending on the assembly, 62-100% of the transformants contained a recombinant plasmid. This indicates that only two colonies had to be picked to ensure 100% correct inserts . T hese new vectors allow for a new faster cloning strategy that can be used for automated cloning platforms.

New chassis strains for heterologous gene expression
We pr e viousl y r eported that deletion of the EYK1 gene impairs the ability of the yeast strain to metabolize erythritol, and in that deleted strain, erythritol could be used as a free inducer. In addition, in the eyk1 strain, erythritol-inducible promoters present higher expression and induction levels (Trassaert et al. 2017 ). Ther efor e, the EYK1 deletion was introduced into JMY1212 together with the introduction of a LYS5 deletion to introduce lysine auxotrophy via successive gene deletion and marker rescue, r esulting in str ain JMY7126 ( Fig. 1 ; P ark et al. 2019b ). Since we observed filamentation of rProt producing strains of Y. lipolytica during fed batch fermentation at high cell density, resulting in Table 4. Expr ession v ectors constructed during this study. Vectors contain the RFP or the AmilCP gene to produce the c hr omopr otein r esulting in r ed or blue E. coli colonies, r espectiv el y. The suffix '-ex' for the marker indicates the presence of Lo xR/Lo xP motifs that are excisable using a Cre-lox recombination method (Fickers et al. 2003 ).

Promoter
Characteristics Constitutive  red  blue  red  pHp4d Phase-dependent, 4 copies UASxpr2-coreLEU2 red blue red pHU8EYK Erythritol-inducible, 8 copies UASxpr2-coreEYK1 red blue red pEYK1-3AB Erythritol-inducible, 3 copies UASeyk1-pEYK1 red blue red pEYL1 Erythritol-inducible red blue red pEYL1-5AB Erythritol-inducible, 5 copies UASeyk1-pEYL1 red blue red partial cell lysis that ensured partial rProt degradation, which affected the quality of the Y. lipolytica secretome (Nicaud et al. unpublished), we aimed to introduce a mutation to prevent the dimor phic switc h. Se v er al genes hav e been shown to abolish the filamentation upon inactivation. Among these genes, only the mutation in MHY1 (YALI0B21582g), coding for the C2H2-type zinc finger protein Mhy1p required for dimorphic transition, results in a strain that did not exhibit hyphae formation under various culture conditions (Konzock and Norbeck 2020 ). A weak positive effect on lipid accumulation and few detectable negative side effects on growth and stress tolerances were observed, although the effect on protein production was not reported. T herefore , to further impr ov e our JMY7126 rPr ot r ecipient str ain, we intr oduced the MHY1 deletion in se v er al rPr ot pr oducing str ains to abolish the filamentation switch of Y. lipolytica . This deletion did not significantl y affect gr owth while the le v el and quality of the secreted pr otein was impr ov ed (Nicaud et al. unpublished). As an example, deletion of MHY1 gr eatl y impr ov ed pr oduction le v els of avian defensin AvBD2 and AvBD7 (Vidal, Lalmanach, Nicaud et al. to be published). Consequentl y, we intr oduced an MHY1-deletion, by tr ansforming JMY7126 with GGE0440, a CRISPR-Cas9-LYS5 ex-MHY1 plasmid, upon selection of Lys + Fil − transformants (Fig. 3 A). The re plicati ve CRISPR-Cas9-LYS5 ex-MHY1 vector was cured through successiv e gr owth on YPD media and a Lys − Fil − clone, JMY8647, was k e pt as the ne w r ecipient str ain. The ne w str ain is unable to filament, displaying smooth colonies compared to the mother strain on the rich media agar plate (Fig. 3 B). In liquid YPD, the mother strain JMY7126 forms ovoid and filament cells (Fig. 3 C) whereas the new strain clearly forms only ovoid cells (Fig. 3 D).

Expression str a tegies for the cloning and secretion of ROL
The ROL lipase from Rhizopus oryzae has a structure homologous to that of the Y. lipolytica extracellular lipase Lip2. Ho w ever, their amino sequences differ, mainly in the pre-and pro-regions, as shown in Figure S2 (Supporting Information). The 392 amino acid (AA) sequence of ROL contained a long 26 AA pre-sequence, follo w ed b y a 69 AA pro-sequence that ended with a KR motif follo w ed b y the 297 AA matur e form. In contr ast, the Y. lipol ytica 334 AA Lip2 contained a shorter 13 AA pre-sequence follo w ed b y four XA/XP dipeptides, and a short 12 AA pro-sequence that ended with a KR motif follo w ed b y the 301 AA mature form (Celi ńska et al. 2018 ). The ROL contained six cysteines involved in three C-C bridges in the mature form: C57-C296; C68-C71; C263-C272. In contrast, Lip2 contained nine cysteines, of which eight are involved in four C-C bridges in the mature form: C30-C299; C43-C47; C120-C123; C265-C273. The free cysteine 244 was changed into an alanine resulting in a thermostable enzyme (Bordes et al. 2011 ). ROL contained four putative N-glycosylation sites, with one in the pr o-r egion N88LT (number ed fr om the ATG) and three in the mature form N124FS , N197LS , and N210PT (number ed fr om the matur e form), while Y. lipol ytica Lip2 contained two N-gl ycosylation sites, N113IS and N134NT in the matur e form, that hav e been shown to be glycosylated (Jolivet et al. 2007 ). Jolivet and co w orkers have reported that the mutation S115V resulted in an active enzyme while the T136V and N134Q m utants wer e inactiv e. In contr ast, a r eport by Aloulou (Aloulou et al. 2013 ) sho w ed the Lip2 v ariants, expr essed in Pic hia pastoris , wer e activ e and pr esented differ ent temper atur e inactiv ation le v els: N113Q (425 U/mL, low inactivation), N134Q (1125 U/mL, higher activity and reduced inactivation) and N113Q/N134Q (322 U/mL, higher rate of inactivation).
Since ROL and Lip2 hav e pr e-pr o-r egions of differ ent lengths, we sought to determine the influence of the pre-and pro-regions on ROL secretion in Y. lipolytica . We designed eight different constructs, as depicted in Fig. 4 : the full ROL gene (pr e-ROL-pr o-R OL-mature-R OL, named R O3); three constructions of pro-ROLmature-ROL using signal sequences previously identified as robust signal sequences (Celi ńska et al. 2018 )-the signal sequence SP1 of the spYALI0B03564 g (RO4), the signal sequence SP4 of spYALI0D06039 g (RO2) and the signal sequence SP6 of spLip2 (RO1); and different targeting sequences of Lip2 for the expression of matur e-ROL or pr o-ROL-matur e-ROL as follows-pr e-Lip2 (SSL1)-mature-R OL (R O7), pre-Lip2 with tw o XA-XA dipeptides (SSL2)-mature-R OL (R O5), pre-Lip2 with tw o XA-XA dipeptides (SSL2)-pr o-ROL-matur e-ROL (RO8), and pr e-Lip2 with four XA/XP dipeptides (SSL4)-pr o-Lip2-matur e-R OL (R O6). The addition of dipeptide XA/XP of Lip2 was shown to be beneficial for the production on Human interferon INF α as reported in (Gasmi et al. 2011 ). All genes were codon optimized according to Y. lipolytica codon bias and with Bam HI and Avr II for cloning purposes (Table S2, Supporting Information). Genes were cloned into the c hr omogenic v ectors JME5599 containing LYS5 ex as marker and pTEF as promoter. The recombinant plasmids were transformed into Y. lipolytica by integration at the docking zeta platform and transformants were selected on YNBD + uracil. Insertion of R OL w as verified b y Y. lipolytica colony PCR. For each assembly, at least 3 independent colonies were k e pt for lipase acti vity tests . T he URA3 fr a gment was then transformed to obtain prototrophic strains (Table 2 ).

Fast screening of lipase production by ROL expressing strains
To e v aluate the lipase production by tr ansformants expr essing ROL with the various targeting sequences, re presentati ve clones (RO1 to RO8) were selected and drop tests were performed on triolein and shea olein plates (Fig. 5 ). Strain JMY329, overexpressing Y. lipolytica Lip2 (Pignède et al. 2000b ), and strain JMY8671, a pr ototr oph deriv ativ e str ain of JMY8647, wer e used as control for a lipase producing and non-producing strain, respectively.  Successive 10-fold dilutions (10 0 -10 −3 ) of cell suspension were spotted onto the lipid containing plates and incubated at 28 • C. Strains JMY329, ov er expr essing Y. lipol ytica Lip2 (positiv e contr ol, PC), and JMY8671, a pr ototr oph deriv ativ e of the recipient JMY8647 (negative control, NC), were used as lipase producing and non-producing strains, respectively. Pictures are from 5 days incubation.
Lipase production can be seen by the halo of hydr ol ysis ar ound the colonies. After only 2 days incubation, halos could be seen around the colonies on triolein media. The positive control, overproducing Lip2, sho w ed better gro wth and lar ger, clear er halos, while the negative control presented reduced growth and no halo. Similar r esults wer e found on shea olein media, although halos could not be observ ed ar ound the colonies expressing ROL (data not shown). After 5 days incubation on triolein plates, growth and lipase pr oduction ar e easier to see (Fig. 5 ). All recombinant ROL str ains pr esent clear halos surrounding the colonies, howe v er with a reduced halo for ROL5 and ROL7. This demonstrates that all r ecombinant ROL str ains ar e pr oducing activ e lipase, as there is no visible halo surrounding the negative control. Ho w ever, the halos were not as large for the positive control, which contains multiple copies of Lip2. On shea olein plates, only tiny halos were visible, while a larger halo is seen for the Lip2 ov er expr essing strain. This could either be due to a lo w er expr ession le v el of ROL in the expressing strains compared to that of the Lip2 overexpr essing str ain (a LIP2 ov er expr essing str ain with m ultiple copies) or due to R OL sho wing better activity to w ar d triolein compared with shea olein. While this fast test confirmed ROL production, it could not allow for the comparison of targeting sequences on the ROL pr oduction le v el. Ther efor e, an activity assay, with p -NPB as substrate, was performed to further evaluate the influence of the differ ent tar geting sequences.

Lipase production depends on targeting sequence
To determine the most efficient targeting sequence for ROL production, the extracellular lipase activities produced were compared after 72 h flask culture in rich YPD media. The supernatant of JMY8671, a pr ototr oph deriv ativ e of the recipient JMY8649, was used as a non-pr oducing contr ol str ain (NC). As shown in Fig. 6 and Table S3 (Supporting Information), specific lipase activities on the p -NPB substr ate wer e v ery similar, about 50 mU per mg of CDW for R O1, R O6 and RO8, whereas about 44 mU per mg of CDW was produced b y R O2, R O3 and R O4. Contrasting this, lo w er specific lipase activities were found for RO5 and RO7 (constructs lacking the pro-ROL, Fig. 4 ), with high variability of lipase activity due to strain instability. These strains displayed slightly reduced growth and loss of the expr ession cassette, likel y due to secretion burden and/or cell toxicity resulting from intracellular lipase activity accumulation. These results also show that signal sequence SP4 (in RO2) and SP1 (in R O4) w er e equall y efficient as the ROL nativ e signal sequence (in the R O3 construct). Ho w e v er, a 23% incr ease of lipase pr oduction was r eac hed using SP6 together with the pr o-r egion of R OL (in the R O1 construct). Using the Lip2 signal sequence and pr o-Lip2 r egion (in RO6), r esulted in a 21% incr ease in specific activity, thus suggesting that the pro-Lip2 region is as efficient as the pr o-ROL r egion for the folding of ROL. In contrast, constructs that lac ked a pr o-r egion r esulted in instable strains and reduced lipase pr oduction. RO1 (SP6-Pr o-ROL-matur e-ROL) showed the highest lipase activity and lipase specific activity and was chosen for the next step of optimization.

Optimization of ROL production
We hav e pr e viousl y demonstr ated that the best promoter for rProt production is not always the strongest one and will depend on the expr essed pr otein (Dulermo et al. 2017 ). To test the mor e efficient pr omoter for ROL pr oduction, the lipase activity le v el was compared using three stronger and/or inducible promoters (Dulermo et al. 2017, Vidal et al. 2023 ) in addition to the constitutive pTEF promoter used abo ve . T he ROL1 gene was subsequently cloned in vectors containing either pHp4d, pHU8EYK or pEYL1-5AB, resulting in plasmids JME5707, JME5708 and JME5709, r espectiv el y ( Table 3 ). The corresponding recombinant plasmids were transformed into JMY8647 to select the mono-copy strain. JMY8647 is a mhy1 deri vati ve of JMY7126. Transformants were selected on solid YNBD media supplemented with lysine or uracil depending on the plasmid selection mark er. Four indi vidual transformants for eac h pr omoter wer e k e pt for lipase production tests . T hen, a second transformation with URA3 or LYS5 genes was performed for the isolation of pr ototr oph clones (Table 2 ).
Lipase specific activities were measured in YNBDE media at 72 h of culture (Fig. 7 ). YNBDE media was used for optimal erythritol-inducible promoter expression. Similar specific lipase activities were obtained for each biological replicate carrying the ROL expressed under the same promoter. As shown in Fig. 7 and in Table S4 (Supporting Information), the mean values of the specific lipase activities were 88.5 ± 11.1 mU/mg CDW for pTEF, 104.8 ± 10.3 mU/mg CDW for pHp4d, 195.7 ± 19.9 mU/mg CDW for pHU8EYK and 183.4 ± 12.2 mU/mg CDW for pEYL1-5AB. Highest lipase production was obtained when using hybrid erythritolinducible promoters. Under these promoters, specific lipase activities were about 1.9 and 2.2 times higher than the pHp4d and pTEF, r espectiv el y.

Multi-copy strain construction to increase lipase production
To further incr ease ROL pr oduction, double-copy ROL expr essing str ains wer e constructed. To obtain these, strain JMY9296 (containing LY S5 ex-pHU8EYK-SP6-Pro-R OL, Lys + Ura − ), sho wing the highest le v el of lipase activity, was transformed with the cassette carrying URA3 ex-pEYL1-5AB-SP6-Pro-ROL to isolate transformants containing two copies of the ROL expression cassette (Table 2 ). Nine transformants were selected and lipase production was determined ( Fig. 8 and Table S5 (Supporting Information)).
Lipase specific activities were measured in YNBDE media at 72 h and compared with that of both the mono-copy JMY9308 (pHU8EYK-ROL) and the pr e viousl y constructed monocopy JMY9147 (pTEF-ROL). The 9 strains containing a second ROL expression cassette showed significantly higher lipase specific activity (mean of 266.7 ± 19.4 mU/mg CDW ), about 1.4 times higher than the mono-copy strain containing pHU8EYK-ROL (185.5 ± 19.4 mU/mg CDW ) and about 3.3 times higher ROL expressed than under the pTEF promoter (79.7 ± 12.3 mU/mg CDW ).

ROL enzyme production level
To assess the pr otein pr oduction le v el and purity, supernatants of the multi-copy strains were compared with that of the mono-copy strains containing pHU8EYK-R OL or pTEF-R OL. SDS-PAGE analysis sho w ed that the m ulti-copy str ains pr oduced mor e pr otein than the mono-copy strain (Fig. 9 ). It also highlighted that ROL is the main secr eted pr otein in those conditions . T he main band, migr ating at ar ound 30 kDa, corr esponds to the expected size of 32.349 kDa of the mature ROL form. Protein concentration estimated using bovine serum albumin calibration curve indicated that ROL r epr esented about 2 g/L.

Discussion and conclusion
The de v elopment of rPr ot pr oduction is based mainl y on the establishment of an efficient expression system and an improved fermentation process . T his ma y in volve the design of an easy Figure 6. Lipase production depending on targeting sequence used. (A) Schematic representation of the expression cassette containing the zeta region for c hr omosomal integr ation, the LYS5 marker, the constitutiv e pTEF pr omoter, the matur e ROL, the Lip2 terminator, and with the differ ent tar geting sequences (SP(x)) tested. (B) Specific lipase activity. Lipase activity in the supernatant was measured after 72 h culture in YPD at 28 • C. JMY8671, a pr ototr oph deriv ativ e of the recipient JMY8649, was used as non-producing control strain (negative control, NC). Results are from three independent clones. SEM values are provided. RO order is identical to Fig. 4 . containing ROL under the promoters; pHp4d (orange), pHU8EYK (blue) and pEYL1-5AB (purple). Strain JMY9147 with ROL under the pTEF promoter (green) was used for comparison and strain JMY8671 was used as negative control (NC, grey). Cells were grown in the inducible YNBDE media for 72 h at 28 • C. Specific lipase activities were measured in triplicate. SEM values are provided.
cloning system and an optimized chassis host strain, an issue specificall y addr essed in this r eport. The ne w set of cloning v ectors is based on pr e viousl y designed auto-cloning vectors, allowing the insertion of the gene of interest at a multi-cloning site and the integration of the expression cassette at a zeta-docking plat-form (Pignède et al. 2000b ). The expression cassette, r eleased fr om the v ector upon Not I digestion, is ther efor e free of bacterial DNA and marker. To facilitate cloning of the gene of interest, a chromogenic marker was inserted at the cloning site, thus enabling the identification of the recombinant plasmid and Specific lipase activity of ROL of nine independent m ulti-copy tr ansformants containing ROL under the inducible pr omoters pHU8EYK and pEYL1-5AB (blue-pur ple), compar ed to the mono-copy str ain JMY9308 (pHU8EYK, blue), and to JMY9147 (pTEF, green). Strain JMY8671 was used as negative control (NC, grey). Cells were grown in the inducible YNBDE media for 72 h at 28 • C. Specific lipase activities were measured in triplicate. SEM values are provided. allowing the use of a faster cloning protocol to reduce the number of cloning steps and the cloning time to less than one week.
The lack of an effective inducible gene expression system has been a major hurdle in making Y. lipolytica a competitive host for rPr ot pr oduction. The most efficient inducible promoters used pr e viousl y in Y. lipol ytica wer e the pLIP2 and pPOX2 promoters, which could be induced by oleic acid (Nicaud et al. 2002, Sassi et al. 2016 ). These are disadvantaged by oleic emulsion impairing easy measurement of growth. The new vector set is based on the recently designed hybrid erythritol-inducible promoters (Trassaert et al. 2017, Park et al. 2019a, Vidal et al. 2023. The ne wl y de v eloped vector set contains the most used auxotrophic markers URA3 , LEU2 , and LYS5 for the selection of recombinant strains. In conclusion, the new vector set will allow fast, easy cloning of a gene of interest under the pTEF constitutive promoter for gene comparison (for example in the e v aluation of differ ent enzymes or differ ent targeting sequences), and under hybrid erythritol-inducible promoters for optimal rProt production. The use of a chromogenic system opens the possibility of adapting this cloning system for high throughput automated cloning platforms.
The main publicly available Y. lipolytica host strains have been r e vie w ed b y Madzak (Madzak 2021 ). The most used str ains ar e P o1d, P o1 g and Po1f, derived from the wild-type French strain W29 (ATCC 20460, Clib 89) (Nicaud et al. 2002 ). Indeed, the Po1 g strain is available in the Y. lipolytica expression kit YLEX, commercialized by Yeastern Biotech. This strain retains only the leucine auxotrophy, allowing its transformants to be prototroph upon transformation with an expression cassette containing the LEU2 marker. Ho w e v er, this str ain is not suitable for rProt production at industrial scale since it contains a pBR322 doc king platform. Curr entl y, the most fr equentl y used host for genetic engineering or protein expression is Po1f, a leucine and uracil auxotroph that contains the deletion of the two main proteases; the alkaline and acid extr acellular pr oteases encoded by XPR2 and AXP , r espectiv el y. Later, the JMY1212 c hassis str ain, deriv ed fr om Po1d, was de v eloped for high-throughput screening of evolved enzymes, which contains the additional deletion of the three main lipases Lip2, Lip7 and Lip8 and a zeta platform that allows the targeted integration of a unique copy of any zeta-based vector  ). This was applied to evolved Y. lipolytica lipases Lip2 (Bordes et al. , 2011 and Candida antarctica lipase B (Emond et al. 2010 ). Mor e r ecentl y, the deriv ativ e JMY7126 was designed to tak e ad v anta ge of the ne wl y de v eloped erythritol-inducible pr omoters obtained by deleting the erythrulose kinase encoded by EYK1 ( eyk1 ) and introducing an additional deletion of the LYS5 gene ( lys5 ) for multiple gene insertion (Soudier et al. 2019, Park et al. 2019a. During submerged fermentation in bioreactors, the process oper ating par ameter could affect the mor phology and rheological behavior of Y. lipolytica (Fillaudeau et al. 2009 ). Indeed, numerous conditions could induce the cells to switc h fr om yeast to filamentous cells (Domínguez et al. 2000 ). This would affect the fermentation parameters and induce cell l ysis, r esulting in pr oteol ytic degradation of the rProt, a drawback also identified during the development of fungal cell factories (Zoglo w ek et al. 2015 , Lübeck andLübeck 2022 ). To skirt this dimor phic switc h in Y. lipol ytica , the MHY1 gene was deleted in the new host strain JMY8647. This new platform contains nine cumulated gene deletions including the mhy1 deletion to pr e v ent filamentation and the eyk1 deletion for optimal hybrid erythritol-inducible promoter utilization, and two auxotrophic markers (uracil and lysine auxotrophies) to intr oduce rPr ot expr ession cassettes.
The r ele v ant industrial lipase ROL fr om Rhizopus oryzae was used as a model protein to assess the efficiency of the new vectors, to identify the most efficient targeting sequence for ROL secretion and to evaluate the best erythritol-inducible promoter for ROL production. Among the robust signal sequences previously identified for rProt production in Y. lipolytica , the SP6 pre sequence w as sho wn to allo w a 23% impr ov ement of ROL pr oduction, whic h is lo w er than the 4 and 6 fold incr eases pr e viousl y r eported for the Thermomyces lanuginosus glucoamylase and the Sitophilus oryzae alpha-am ylase, respecti vely (Celi ńska et al. 2018 ). Ho w ever, the increase is in relation to the production observed using the native pr omoter, whic h may be higher for ROL than for the pr e viousl y r eported pr oteins. Importantl y, we hav e with this work demonstrated that a pro region is required for efficient secretion and strain stability, and that both pro-ROL or pro-Lip2 could be used for ROL production. In contrast, the pro-ROL region was not used for the expression of ROL in Pichia pastoris by Chow and Nguyen (Chow and Nguyen 2022 ), who used the Sacc harom yces cerevisiae pr epr o r egion of the alpha factor. Using the strong methanolinducible AOX1 promoter for ROL production, they could produce wild-type and thermostable variants of ROL at 40 to 120 mg/L in P. pastoris . The use of hybrid erythritol-inducible promoters and the insertion of two expression cassettes resulted in strains with two copies, producing approximately 3.3 times more ROL than the monocopy strain containing the ROL gene under the pTEF promoter, corresponding to about 2 g/L of lipase in the supernatant in our conditions in flask culture . T he production level of ROL in Y. lipolytica is 25 times higher than in P. pastoris . This confirms that Y. lipolytica can perform better than P. pastoris in bioreactor cultures in terms of cell growth, enzyme titer, and production time, for specific enzymes as pr e viousl y shown in the production of Candida antarctica lipase B (Theron et al. 2020 ). This demonstrates that our ne w expr ession system and Y. lipol ytica c hassis str ain could be an attr activ e host for the screening of e volv ed enzymes, to be used for production of structured triacylglycerols (TAGs), i.e. modification of the positional distribution of fatty acids on the gl ycer ol bac kbone, for a pplications in e.g. the food industry.
In conclusion, the new chromogenic vector set and host strain r epr esent an attr activ e platform for enzyme evolution and enzyme production.